Acquired genetic mutations underlie all forms of cancer and are likely to be important in aging. DNA damage, a major cause of mutations, can occur on one DNA strand or both strands; in the latter case, a double-strand DNA break results. The two forms of double-strand break repair in multicellular eukaryotes are homologous recombination (HR) and nonhomologous DNA end joining (NHEJ). This proposal is devoted to understanding the NHEJ pathway at the biochemical and genetic levels. The NHEJ pathway is the major pathway for repairing double-strand DNA breaks during G0, G1, and early S phases of the cell cycle; therefore, the understanding of NHEJ is of broad medical importance. The major effort of this proposal is to understand the biochemistry of key steps in the NHEJ pathway and to reconstitute the pathway with biochemically purified proteins. In Specific Aim 1, we test four hypotheses related to the earliest steps in NHEJ. In the first, we examine whether Ku recruits the Artemis:DNA-PKcs complex to a DNA end. In the second, we examine the point at which inositol phosphates might regulate NHEJ. In the third, we examine how protein phosphorylation affects the initial complex of proteins that are thought to bind at a broken DNA end. In the fourth, we use a murine mouse model to test for interference between proteins in the early phase of the NHEJ pathway. In Specific Aim 2, we examine how two broken DNA ends are brought into physicial proximity. Specifically, we test whether this can be achieved by the Ku:Artemis:DNAPKcs complex. In Specific Aim 3, we describe efforts to reconstitute the entire human NHEJ pathway using purified proteins. As part of this aim, we also test for roles of DNA polymerases, which are the one type of enzymatic activity yet to be definitively determined for NHEJ in higher eukaryotes. Specific Aim 4 is directed at defining the active site for the major nuclease in the NHEJ pathway, Artemis. This aim is also directed at understanding the region of interaction between Artemis and DNA-PKcs. Overall, this proposal represents a major concerted effort to deepen and complete our understanding of this primary pathway of repairing double-strand DNA breaks. The long-term medical benefit of a biochemically-defined NHEJ system includes the ability to test small molecule drug inhibitors for roles in cancer therapy. [unreadable] [unreadable]